Molecular Mechanism Research On Rice Seed Longevity With Antisense LOX-3 Gene

Research on rice seed longevity is enjoying unprecedented growth as pressure looms for global rice market to meet an ever increasing demand for rice supply accompanying with serious loss during rice storage.Deterioration of rice grains and the development of stale flavor during storage are serious problems which reduce the quality of stored rice.Lipoxygenase(LOX)is a kind of dioxygenase containing non-heme iron,which catalyzes the peroxidation of lipids in rice grains,leading to the formation of volatile compounds.There are three kinds of lipoxygenase isozymes in rice embryo,LOX-1,LOX-2 and LOX-3.LOX-3 is the most major with the highest component.In this study,we obtained transgenic plants with antisense expressing of LOX-3 gene,then through the analysis and identification of transgenic plants to explore the relationship between LOX-3 and rice seed longevity.The main results are as follow:1.The plasmids(pHG1PL,pHG4PL and pHG4P500L)containing LOX-3 antisense gene were introduced into indica rice restorer line Hang 1 and Hang 2 mediated with Agrobacterium transformation.The results of PCR and Southern blot analysis indicated that the target gene of antisense LOX-3 had been integrated into the rice genome.Compared the expression level of embryo LOX-3 in transgenic rice and control,found that LOX-3 expression was significantly decreased in transgenic rice endosperm,which showed that the introduced of antisense LOX-3 gene into rice endosperm has successfully inhibited the expression of LOX-3.2.Investigated the phenotype of transgenic rice and control,including plant height,tiller number,seed setting rate and 1000-grain weight,found that there were no significant differences between transgenic rice and control,except for transgenic line No.63,its tiller number and seed setting rate were significantly different from the control,indicating that the inhibition of LOX-3 expression in rice endosperm did not make significant impact on their characteristics.3.Using KI-I2-starch coloration method to detect the activity of LOX-3 in transgenic rice,compared the differences of the color between different reaction solution and found that the control was dark,the LOX-3 deletion mutant DawDam was colorless,while the transgenic plants were light red,suggesting that the activity of LOX-3 in transgenic rice embryo was decreased.Then detected the content of linoleic acid in transgenic rice seed and control using high performance liquid chromatography(HPLC),to find that the content of linoleic acid in transgenic rice is much higher than the control and the difference was significant,the result further confirmed that the introducing of antisense LOX-3 gene into transgenic rice endosperm had successfully reduced the activity of LOX-3.4.Compared the differences of storability between transgenic rice seeds and control with artificial aging and natural aging,and found that the germination ratio of them both declined during these two different treatments,and the declining range was larger and larger with increasing of the aging time.While the germination rate of transgenic rice seeds was significantly higher than control with the same treatment.These results showed that the longevity of rice seed had been increased by reducing the activity of LOX-3 in rice endosperm.5.Using the scanning electron microscope to see the shape of the starches in transgenic rice seeds and control before and after artificial aging,found that the starches of transgenic rice seeds were in the same size and tightly packed before aging,while they were not very uniform and a little loose in the seeds of control.After artificial aging for 30d,some of the starches in transgenic rice seeds turned to be un-uniformed in shape and size,while they changed much more significantly in control.It suggested that through inhibiting the expression of LOX-3 in rice endosperm,the longevity of rice seed had been improved.6.Compared the grain quality of transgenic rice and control,the results showed that the content of fat in the transgenic rice seeds was higher than the control.After artificial aging,the content of fat in them both declined,but it was also higher in the former.The results showed that the inhibition of LOX-3 expression in transgenic rice endosperm did not cause any significant changes of rice quality,instead of increasing the fat content in rice,indicating that through inhibiting the activity of LOX-3 in the rice endosperm can reduce the metabolism of lipids.7.Constructed the expression profile of differential proteins between antisense LOX-3 transgenic rice,control and storable rice varieties Yunhui 290 and Fuhui 7185 before and after artificial aging using iTRAQ markers combined with liquid chromatography and mass spectrometry.Analyzed the function and the metabolic pathway of differential proteins by GO annotation,COG annotation and KEGG metabolic pathway annotation.In order to provide theoretical basis for revealing the role of LOX-3 in rice storage,and also provide reference on further exploring the molecular mechanism of rice seed longevity.8.Compared the differences of marker gene deleting efficiency between two different heat shock methods,and found that the efficiency was much higher when treating with transgenic plants at four-leaf stage.It was 83.3%.At last,confirmed an appropriate method for marker gene deleting in rice to obtain transgenic rice without selective marker and improve the biological safety and food safety of transgenic rice.